Effective new cancer therapies have been developed based on agents with novel mechanisms of action, and those that target the pathophysiology of malignances. Clinically active in phase I studies in relapsed/refractory acute myeloid leukemia and myelodysplastic syndromes, Sapacitabine, the orally bioavailable form of 2'-C- cyano-2'-deoxy-1-2-D-arabino-pentofuranosylcytosine (CNDAC), acts by the unique mechanism of action of causing a single strand break in DNA. In the process, the incorporated analog is converted to a chain- terminating dideoxynucleotide, CNddC, a nucleotide that cannot be extended. Importantly, our initial results demonstrated that these nicks in DNA are relatively stable, and that they are converted to one-ended double- strand breaks when cells attempt to replicate their DNA across the nick. Further, our studies suggest that cells are mainly dependent upon homologous recombination (HR) to repair double-strand breaks that arise at collapsed replication forks. Thus, strategies that increase CNDAC incorporation into DNA, or approaches that diminish expression of HR pathway proteins would likely be synergistic with CNDAC. A corollary is that diseases with inherent deficiencies in homologous recombination repair would be selectively sensitive to the actions of CNDAC and its congeners. The overall goal of our proposal is to develop a thorough understanding of the mechanism by which CNDAC causes cell death, and to investigate strategies that will extend its toxicity to quiescent cells, overcome resistance due to highly expressed homologous recombination proteins, and provide rationale for the design combination strategies for translation to the design of clinical trials. We hypothesize that answers to questions posed in the following specific aims will clarify the mechanism of CNDAC action and indicate strategies to direct its application in the clinic. 1. What DNA damage repair mechanisms recognize and process CNDAC-induced DNA damage? By what mechanism is the CNddC lesion excised from DNA? Can mechanistic interactions be identified to provide a rationale for the design of combinations with CNDAC? By what mechanisms do histone deacetylase inhibitors (HDACi) sensitize cells to the action of CNDAC? PUBLIC HEALTH RELEVANCE: This application proposes to develop a thorough understanding of the mechanism by which the small molecule CNDAC and related compounds cause cell death, and to investigate how tumors respond to avoid being killed. This information will be used to develop rationales for combinations with agents targeted at inhibiting such potential resistance mechanisms for translation to the design of clinical trials. Rationales for mechanism-based combinations with active anti-cancer drugs, and with the epigenetic regulatory pathways for DNA repair protein expression will be developed with a view to broadening the potential clinical application and impact of CNDAC (Sapacitabine).